Phosphorylation of the porcine skeletal and cardiac muscle sarcoplasmic reticulum ryanodine receptor.

Porcine skeletal and cardiac muscle sarcoplasmic reticulum (SR) vesicle fractions enriched in the ryanodine receptor were phosphorylated in the presence of [gamma-32P]MgATP and either exogenous cAMP-dependent protein kinase (cAMP-PK), or Ca2+ plus calmodulin. Phosphorylation of the cardiac muscle ryanodine receptor in the presence of either cAMP-PK or calmodulin (6.4 and 10.6 pmol Pi/mg SR respectively) was approximately equal to or twice the [3H]ryanodine binding activity of this preparation (5.2 pmol/mg). Furthermore, cardiac muscle ryanodine receptor Pi incorporation catalyzed by cAMP-PK and calmodulin was approximately additive. In skeletal muscle SR, however, the level of cAMP-PK or calmodulin catalyzed phosphorylation of the intact ryanodine receptor (0.2 or 2.9 pmol Pi/mg SR, respectively) was much less than the [3H]ryanodine binding activity of this fraction (11.6 pmol/mg). Furthermore, Pi incorporation into the intact skeletal muscle ryanodine receptor was 3-8-fold less than that incorporated into a component of slightly lower M(r). Although this latter component comigrated with an immunoreactive fragment of the ryanodine receptor on polyacrylamide gels, it did not appear to be derived from the ryanodine receptor. We conclude that the significant phosphorylation of the cardiac muscle SR ryanodine receptor indicates a likely physiological role for protein kinase-mediated regulation of this Ca(2+)-channel. In contrast, the minimal phosphorylation of the skeletal muscle SR ryanodine receptor indicates that such a role of protein kinases is unlikely in this tissue.

Ryanodine receptor in different malignant hyperthermia-susceptible porcine muscles.

The sarcoplasmic reticulum (SR) ryanodine receptor was studied in SR vesicles isolated from the vastus intermedius skeletal muscle and cardiac muscle of malignant hyperthermia-susceptible (MHS) and normal pigs. MHS and normal heavy SR preparations isolated from the vastus intermedius muscle had similar yields, polyacrylamide gel electrophoretic patterns, Ca2(+)-ATPase activities, mitochondrial enzyme activities, calsequestrin contents, and maximal [3H]ryanodine-binding activities. However, while half-maximal calcium concentrations (Ca0.5) for stimulation of MHS and normal vastus intermedius SR [3H]ryanodine binding were not significantly different, the Ca0.5 for inhibition of [3H]ryanodine binding to MHS vastus intermedius SR (76 +/- 17 microM) was significantly greater than to normal SR (16 +/- 9 microM). MHS vastus intermedius SR also exhibited a significantly lower Kd value (62 +/- 15 nM) for [3H]ryanodine binding compared with normal SR (Kd = 284 +/- 102 nM). These values for MHS and normal vastus intermedius SR are similar to those reported using SR isolated from a muscle composed of predominantly fast-twitch fibers, indicating the similarity of the ryanodine receptor in fast- and slow-twitch skeletal muscles. In contrast, there were no differences in the properties of the ryanodine receptor of porcine cardiac SR isolated from MHS and normal pigs. We therefore conclude that there is a defect in the SR ryanodine receptor of both slow- and fast-twitch skeletal muscle fiber types but not in cardiac muscle of MHS individuals.

A clinically applicable method to preserve urine and bladder washing cells for flow cytometric monitoring of bladder cancer patients.

We describe a method to fix exfoliated bladder cells that is suitable for followup of bladder cancer patients by deoxyribonucleic acid flow cytometry. After fixation with room temperature methanol plus acetic acid (20:1, volume:volume) urine and bladder washing samples from these patients can be stored at room temperature for 3 to 7 days and then assessed reliably for the presence of aneuploidy and the percentage of hyperdiploid cells. For those with active transitional cell carcinoma diagnostic accuracy comparing fresh to fixed specimens was improved from 58 to 92% with urine and from 50 to 100% with washing samples. For patients with a history of transitional cell carcinoma who currently are free of disease the false positive rate remains unchanged after fixation. The procedure described is suitable for use in the outpatient clinic and should permit shipping of samples without refrigeration to a central flow cytometry facility for analysis.

Deoxyribonucleic acid flow cytometry of benign prostatic disease.

Deoxyribonucleic acid flow cytometry was performed on aspirated prostatic cells from 198 patients who had benign cytological or histological findings. Unsatisfactory acellular histograms were obtained from 10.6 per cent of the cases. Three-fourths of the satisfactory samples (more than 5,000 cells after subtracting debris) showed the expected single peak deoxyribonucleic acid diploid to near diploid histograms. Unexpectedly, the remaining samples were deoxyribonucleic acid aneuploid, most having 2 peridiploid peaks (deoxyribonucleic acid index 0.82 to 1.31). Usually, proliferation was low with less than 20 per cent hyperdiploid cells and with 2.5 +/- 1.5 per cent G2 cells. In 10 per cent of the single peak histograms there was evidence of inflammation, identified as an increase in hyperdiploid cells without an increased percentage of G2 cells but with a tail of high channel values. The aforementioned histogram features were considered to be benign findings. Seven per cent of the samples had deoxyribonucleic acid histograms suggestive of prostate cancer. Of these samples 7 had diploid or peridiploid aneuploid histograms with high proliferation (more than 20 per cent hyperdiploid cells with 8.5 +/- 3.8 per cent G2 cells), while 5 had histograms with deoxyribonucleic acid aneuploidy other than peridiploidy.

Characterization of urinary keratin number 18 using a new assay.

The levels of the low-molecular weight keratin 18 appearing in the voided urine of 130 healthy volunteers was studied using a new immunoradiometric assay for keratin 18 (IRMAK-18). Keratin 18 was detected in 79 urines with an average level of 2.5 +/- 4.7 ng./ml. The assay does not detect complex epidermal keratins or non-keratin intermediate filaments. Only 50% of the purified keratin added to urine could be recovered. The recovered keratin had the same molecular weight as the input molecules, implying that substances in the urine physically inhibited the assay rather than destroyed the keratin. The independent variables of cell count, dead cells, and protein concentration were not found to be important in evaluating the results. However, the urine concentration, as measured by creatinine, might be an important consideration for assessing the levels of keratin detected.

Urine: a suitable sample for deoxyribonucleic acid flow cytometry studies in patients with bladder cancer.

Deoxyribonucleic acid flow cytometry of bladder washings has proved to be a valuable procedure for the diagnosis and followup of patients with transitional cell carcinoma. However, for this procedure to gain maximal acceptance, it should be possible to use voided urine specimens instead of bladder washings. To evaluate this possibility we compared histogram findings for 114 bladder washings and 122 concomitantly obtained urine samples (voided and catheterized) from 89 consecutive patients who had active or a history of transitional cell carcinoma. Unsatisfactory histograms were obtained in 4 per cent of the urine samples and in 2.6 per cent of the bladder washing samples. The satisfactory rate for voided or catheterized urine samples was the same. We conclude that in this patient population satisfactory deoxyribonucleic acid histograms can be obtained from samples of voided urine.

Softening of thermoplastic polyurethanes: a structure/property study.

Softening of thermoplastic polyurethanes (TPU) in a simulated body environment (37 degrees C n-saline) was studied as a function of composition, structure and resultant morphology of these (AB)n type block copolymers. The structural variations were attempted by changing chemical composition and molecular weight of both hard A and soft B segments and their weight ratio in the polymer. In addition, the influence of bulk and/or surface modifiers, such as "reacted-in" polysiloxanes and fluorinated polyalkylether glycols, was also investigated. The degree of softening, expressed as a percentage decrease of the elastic modulus (5% tensile modulus) upon two hours exposure to the testing environment, is significant, reversible and depends on the ratio of hard to soft segment and the extent of microphase separation. Since these parameters can be selected during the polymer synthesis and processing into desirable shapes, the degree of softening can thus be controlled. This softening at body temperature represents one of the most notable performance advantages of these biomaterials.